KR20170013073A - Quantitative Analysis for Oligomers in Polymer using MALDI-TOF Mass analysis - Google Patents

Abstract

The present invention provides a guanidine group which was substituted and labeled as ^13C, or a guanidine-based polymer which comprises a biguanidine group. The present invention relates to a quantitative analysis, based on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, of an amount of an oligo-[2-(2-ethoxy)-ethoxyethyl]-guanidinium chloride (PGH), a polyhexamethylene guanidine (PHMG) oligomer, and a polyhexamethylene biguanide (PHMB) in the guanidine-based polymer included in household goods.

Description

[0002] Quantitative analysis of oligomers in polymers using MALDI-TOF mass spectrometry [

The present invention relates to a method for quantitative analysis of oligomers in polymers using MALDI-TOF mass spectrometry, and more particularly, to a method for quantitative determination of oligomers in a polymer by MALDI-TOF mass spectrometry using a radioisotope- Analysis method.

Guanidine-based disinfectants such as oligo- [2- (2-ethoxy) -ethoxyeth-yl) -guanidinium-chlorid, PHMG (polyhexamethyleneguanidine ephosphate) and PHMB (polyhexamethylene biguanide) Has been widely used. On the other hand, since they are not biodegradable, environmental pollution is a concern, and aquatic toxicity may be serious, and various problems such as being highly toxic even at 10 μg / L (0.000001%, 0.054 μM, 0.01 ppm) In addition, it is prohibited to use it as a spray because of its toxicity to inhalation. However, it can be very dangerous if it is manufactured and used as an arbitrary, indiscriminate application without regulation.

Among them, PGH (Oligo- [2- (2-ethoxy) -ethoxyethyl] -Guanidinium Chloride) and PHMG (Polyhexamethylene Guanidine) have been used as humidifier disinfectants in Korea and dozens of people died in 2011, .

Since these components disrupt the phospholipid bilayer and show a sterilizing effect, there is a possibility of destruction of cell phospholipids in humans and there is a fear of destruction of skin barrier. Therefore, although it is possible to cause dermatitis if it is frequently used in skin such as a wet tissue, it can still be used extensively in living environment products such as disinfectants, preservatives, deodorants and wet tissues. In order to prevent abuse of these components, In particular, there is a need for the development of simple and quantitative detection methods for these oligomer components.

In this regard, Korean Patent No. 10-1503638 (Feb. 17, 2015) discloses a reagent which reacts with a disinfectant (PGH, PHMG and PHMB) to cause a color reaction, A method for detecting a harmful fungicide component in a living environment product, and a detection kit are disclosed. However, the preceding literature has a problem that quantitative analysis can not be performed, only the qualitative detection of the presence or absence of PGH, PHMG and PHMB is possible.

On the other hand, MAIDI-TOF (Matrix-assisted laser desorption / ionization time-of-flight) method is used as a method of analyzing a polymer substance.

The MAlDI-TOF mass spectrometer mixes a sample to be analyzed with an excessive matrix on a sample plate, and then, the sample / matrix mixture is dried to form a crystal structure. The sample plate is placed in a vacuum mass spectrometer, It irradiates ultraviolet or infrared laser to the sample, ionizes or gasifies the sample, discharges the sample to a vacuum measuring instrument for flight time measurement, and measures the mass by measuring the flight time from the vacuum to the detector. In general, a light molecule can be measured at a fast detection time, and the mass of a sample can be measured using the principle that a heavy molecule is detected at a slow detection time.

Japanese Patent Registration No. 4141834 (Aug. 27, 2008) discloses a method for assaying MAIDI-TOF mass spectrometry using a matrix-assisted laser desorption ionization mass spectrometry ICIER) is a technique for providing MAIDI-MS ionization enhancement, relative quantification, and additional database search conditions simultaneously without further sample manipulation using arginine-containing cysteine-modifying compounds useful for MAIDI-MS analysis of proteins However, there is a problem that the internal reference material is prepared as two reagents for labeling the protein for analysis.

Therefore, for the analysis of harmful substances such as guanidine-based disinfectants contained in daily necessities, there is a continuing need to develop a new method for detecting the content of oligomers in a polymer and a sample composition for analysis of a new composition required for the method Is required.

Korean Registered Patent No. 10-1503638 (March 17, 2015). Japanese Patent No. 4141834 (2008.08.27.)

Accordingly, the present invention provides a method for quantitatively analyzing oligomers in polymers such as guanidine-based disinfectants contained in many daily necessities such as wet tissues, hand cleaners, contact lens cleaning liquids, and cosmetic removers.

The present invention also provides a novel composition for a standard material used for the analysis of oligomers in the polymer.

The present invention provides a guanidine based polymer comprising a guanidine group or a biguanidine group labeled with a carbon linked to three nitrogen atoms in a guanidine group substituted with ^13C .

The present invention also relates to a guanidine polymer comprising a guanidine group or a biguanidine group, wherein an alkylene group having 1 to 12 carbon atoms is bonded to any one nitrogen atom in the guanidine group, or a nitrogen atom Wherein at least one of the carbon atoms in the alkylene group is substituted with ¹³ C to provide a guanidine based polymer.

In addition, the present invention provides a ¹³ C represented by a labeled guanidine salt and the structural formula B-1 obtained by the reaction of hexamethylene diamine, ¹³ C-labeled polyhexamethylene guanidine (PHMG) polymer represented by the following structural formulas A-1 do.

Structural Formula A-1 Structural Formula B-1

X in the structural formula A-1 is a halogen or a monovalent anion.

In addition, the present invention relates to structural formulas A-2 to the structural formula A-4 or more of any of the ¹³ C is represented by a labeled guanidine salt and the structural formula B-1 obtained by the reaction of hexamethylene diamine, ¹³ C-labeled polyhexamethylene acetabulum 0.0 > (PHMB) < / RTI >

Structure A-2

Structure A-3

  Structural Formula A-4 Structural Formula B-1

X in the structural formulas A-2 to A-4 is a halogen or a monovalent anion.

The present invention relates to the structural formula A-5 to A-6 obtained by any one of the compounds with the structural formula ¹³ C-labeled hexamethylenediamine salt of the reaction represented by the B-2 is selected from, ¹³ C-labeled polyhexamethylene biguanides (PHMB) polymer.

Structural Formula A-5 Structural Formula B-2

Structure A-6 Structure B-2

Provided that X in the structural formula B-2 is a halogen or a monovalent anion.

The present invention also provides a composition for an internal standard material for analyzing the oligomer content of a guanidine polymer in a household article, comprising the ¹³ C-labeled guanidine polymer.

The present invention also provides a method for analyzing the oligomer content of a guanidine polymer in a household product through a mass spectrometry method using the composition for internal standard.

Further, the present invention provides a method for preparing a sample, comprising the steps of: a) extracting a sample solution containing an oligomer of a guanidine polymer from a household article; b) A sample for the internal reference material containing the ¹³ C-labeled guanidine polymer is mixed into the extracted sample solution, and the sample is prepared by changing the relative concentration of the sample solution and the composition for the internal standard material. Obtaining a MAlDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry spectrum; And c) quantitatively analyzing the oligomer content of the guanidine polymer in the sample solution by obtaining a calibration curve using each of the mass spectrometry spectra, and analyzing the oligomer content of the guanidine polymer in the household article ≪ / RTI >

The present invention also provides a method for analyzing the oligomer content of a polymer included in a detection target, comprising the steps of: a) extracting a sample solution containing an oligomer of a polymer polymer from a detection target; b) preparing a sample by varying the relative concentration of the sample solution and the composition for the internal standard material by mixing the extracted sample solution with a composition for internal standard labeled with a radioisotope, -assisted laser desorption / ionization time-of-flight mass spectrometry spectra; And a step of quantitatively analyzing the oligomer content of the polymer polymer in the sample solution by obtaining a calibration curve using each of the mass spectrometry spectra and analyzing the oligomer content of the polymer polymer contained in the detection target to provide.

The present invention can provide a novel method for analyzing the oligomer content of polymeric polymers contained within a detection subject.

As an example of the analysis method according to the present invention, there is provided a novel method capable of quantitatively analyzing oligomers in a polymer such as a guanidine fungicide contained in many household goods such as a wet tissue, a hand cleaner, a contact lens cleaning liquid, and a cosmetic remover.

The present invention also provides a novel guanidine polymer labeled with ¹³ C used for the analysis of oligomers in the polymer, and a composition for a standard material containing the same.

According to the present invention, when the MAlDI-TOF mass spectrometry is used, the content of the oligomer in the polymer can be quantitatively analyzed to a minimum of 1 ppm by using the composition for ¹³ C-labeled reference material.

Figure 1 is a MAlDI-TOF analytical spectrum of PHMG oligomer isomers.
Figure 2 is the MAlDI-TOF analysis spectrum of the PHMB oligomer isomers.
FIG. 3 is a ¹ H-NMR spectrum of ¹³ C-labeled PHMG. FIG.
4 is a ¹ H-NMR spectrum of ¹³ C-labeled PHMB.
5 is a figure showing a ¹³ C-NMR spectrum of the PHMG ¹³ C labeled.
6 is a figure illustrating a ¹³ C-NMR spectrum of the ¹³ C labeled PHMB.
FIG. 7 is a diagram showing an MAlDI-TOF analysis spectrum of ¹³ C-labeled PHMG synthesized with an internal standard material. FIG.
Fig. 8 is a diagram showing MAIDI-TOF analysis spectrum of ¹³ C-labeled PHMB synthesized with an internal standard material.
FIG. 9 is a graph showing MAIDI-TOF analysis spectra of ¹³ C-labeled PHMG concentration. FIG.
FIG. 10 is a graph showing MAIDI-TOF analysis spectra of ¹³ C-labeled PHMB by concentration. FIG.
11 is a PHMG linear calibration curve obtained using an internal standard material according to Example 1 of the present invention.
12 is a PHMB linear calibration curve obtained using an internal standard material according to Example 2 of the present invention.
13 is an MAIDI-TOF analysis spectrum for each type of solid-phase extraction cartridge according to Example 3. Fig.
14 is a MAlDI-TOF analysis spectrum of PHMG contained in a wet tissue according to Example 4 of the present invention.
15 is a MAlDI-TOF analysis spectrum of PHMG contained in a wet tissue according to Example 5 of the present invention.
16 is a MAlDI-TOF analysis spectrum of the PHMB contained in the wet tissue according to the sixth embodiment of the present invention.
17 is a MAlDI-TOF analysis spectrum of PHMG contained in the contact lens cleaning liquid according to Example 7 of the present invention.
18 is a MAlDI-TOF analysis spectrum of PHMB contained in the contact lens cleaning liquid according to Example 8 of the present invention.
19 is a MAlDI-TOF analysis spectrum of PHMG contained in a makeup remover according to Example 9 of the present invention.
20 is a MAlDI-TOF analysis spectrum of PHMB contained in the makeup remover according to Example 10 of the present invention.
FIG. 21 is a MAlDI-TOF analysis spectrum of a mixture of a PGH standard solution and a PEG standard solution at a specific concentration.
22 is a MAlDI-TOF analysis spectrum of a mixture of pretreated PGH standard solution and PEG standard solution according to Example 11 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, which illustrate preferred embodiments in which the present invention can be readily practiced by those skilled in the art. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail to avoid obscuring the subject matter of the present invention.

The present invention relates to a guanidine polymer comprising a guanidine group or a biguanidine group labeled with a carbon linked to three nitrogen atoms in a guanidine group and substituted with ¹³ C, and an inner part for analyzing the oligomer content of the guanidine polymer in the household article, The present invention relates to a composition for an internal standard.

Herein, the guanidine group means a neutral or cationized group having a carbon atom and a structure in which three nitrogen atoms are bonded to the carbon atom as shown in the following structural formula 1, Quot; means a neutral or cationized group in which two guanidine groups are linked together as shown.

[Structural formula 1]

[Structural formula 2]

In the present invention, the respective substituents connected to the lower two nitrogen atoms in the structural formula 1 and the structural formula 2 in the guanidine group and the biguanidine group are the same as or different from each other and independently represent a monovalent linkage group such as guanidine group or biguanidine The substituent which can be bonded to the group is not limited and may be possible.

In the present invention, the guanidine polymer means a polymer compound having a repeating unit containing a guanidine group or a biguanidine group as defined above and polymerized to form a polymer. This means that the oligomeric compound .

That is, the guanidine polymer of the present invention is characterized in that at least one of the carbons linked with three nitrogen atoms in the guanidine group or the bi-guanidine group of the structural formula 1 and the structural formula 2 is substituted with ¹³ C and labeled.

In a preferred embodiment of the present invention, the guanidine based polymer may include a repeating unit of any one of the following structural formulas A to D:

[Structural Formula A]

[Structural formula B]

[Structural formula C]

[Structural formula D]

Provided that the repeating unit of each compound in Structural Formulas A to D is 1 to 1000. [

Here, the structural formula A corresponds to a repeating unit for producing an oligo- (ethoxy) ethoxyethyl guanidine polymer (PGH), and the structural formula B corresponds to a repeating unit for producing a polyhexamethylene guanidine (PHMG) polymer The structural formulas C and D correspond to repeating units for producing polyhexamethylenebiguanide (PHMB) polymers.

The present invention also relates to a guanidine polymer comprising a guanidine group or a biguanidine group, wherein an alkylene group having 1 to 12 carbon atoms is bonded to any one nitrogen atom in the guanidine group, An alkylene group having 1 to 12 carbon atoms is connected, and at least one of the carbon atoms in the alkylene group is substituted with ¹³ C to thereby provide a guanidine polymer.

In this case, in the guanidine polymer of the present invention, the carbon contained in the guanidine group or the guanidine group is not substituted with ¹³ C, and any of the alkylene groups connected to nitrogen atoms in the guanidine group as shown in the following Structural Formulas 3 and 4 Characterized in that at least one carbon atom is substituted with ¹³ C and labeled. In this case, the alkylene group may be an alkylene group having 1 to 12 carbon atoms.

[Structural Formula 3]

[Structural Formula 4]

As a more specific example, an oligonucleotide wherein the guanidine-based polymer of the present invention labeled with ¹³ C - [2-ethoxyethyl guanidinium chloride (oligo- [2- (2-ethoxy ) -ethoxyethyl] to (2-) -guanidinium chloride; PGH), labeled with ¹³ C polyhexamethylene guanidine phosphate (Polyhexamethyleneguanidine phosphate) or labeled with ¹³ C polyhexamethylene guanidine hydrochloride (Polyhexamethyleneguanidine hydrochloride) or labeled with ¹³ C polyhexamethylene biguanides hydrochloride (Polyhexamethylene biguanide hydrochloride) or polyhexamethylene biguanidinium phosphate labeled with ¹³ C can be used.

Illustratively, the polyhexamethylene guanidine (PHMG) polymer having the structure B can be prepared by reacting the ¹³ C-labeled guanidine salt represented by the structural formula A-1 and the hexamethylenediamine represented by the structural formula B-1 ≪ / RTI >

Structural Formula A-1 Structural Formula B-1

[Reaction Scheme 1]

X in the structural formula A-1 is a halogen or a monovalent anion.

As another example, the polyhexamethylenebiguanide (PHMB) polymer having the structural formula C or the structural formula D may be prepared by reacting a ¹³ C-labeled guanidine salt of any of structural formulas A-2 to A-4 And hexamethylenediamine represented by the structural formula B-1.

Structural Formula A-2 Structural Formula B-1

Structural Formula A-3 Structural Formula B-1

  Structural Formula A-4 Structural Formula B-1

[Reaction Scheme 2]

X in the structural formulas A-2 to A-4 is a halogen or a monovalent anion.

The present invention also relates to a polyhexamethylenebiguanide (PHMB) polymer represented by the structural formula 4, wherein the carbon contained in the alkylene group connected to the nitrogen atom in the bivalent group is substituted with ¹³ C, ¹³ represented by any one of the compounds with the structural formula B-2 is selected from C-labeled -6 hexamethylene diamine obtained from the reaction salt, and provides a ¹³ C-labeled polyhexamethylene biguanides (PHMB) polymers.

Structural Formula A-5 Structural Formula B-2

Structure A-6 Structure B-2

Provided that X in the structural formula B-2 is a halogen or a monovalent anion.

In the present invention, as a specific example of the monovalent anion in Reaction Scheme 1 or Reaction Scheme 2, the monovalent anion may be an aryloxy anion having 6 to 20 carbon atoms, a carboxy anion having 1 to 20 carbon atoms, an alkoxy anion having 1 to 20 carbon atoms , A carbonate anion having 1 to 20 carbon atoms, an alkylsulfonate anion having 1 to 20 carbon atoms, a cyano group, a nitro group, BF ₄ ^- , ClO ₄ ^- , PF ₆ ^- , and HCO ₃ ^- But is not limited thereto.

Meanwhile, the guanidine group-containing guanidine group or the guanidine group labeled with ¹³ C in the present invention can be used as a composition for an internal standard for analyzing the oligomer content of a guanidine polymer in a household article .

As an embodiment of the present invention, the internal standard composition may be prepared by mass spectrometry, in particular MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry Can be used as a composition for an internal standard material in a method for analyzing the oligomer content of a guanidine polymer in a household article.

Matrix-assisted laser desorption / ionization time-of-flight (MASDI) mass spectrometry has been used as a general method for analyzing polymeric materials through mass spectrometry.

The MALDI-TOF mass spectrometer mixes the sample to be analyzed and the excess matrix on the sample plate, and then the sample / matrix mixture is dried to form a crystal structure. The sample plate is placed in a vacuum mass spectrometer, It irradiates ultraviolet or infrared laser to the sample, ionizes and gasifies the sample, discharges it to the vacuum measurement device for flight time measurement, and measures the mass by measuring the flight time from the vacuum to the detector.

In general, a light molecule is measured at a fast detection time and a heavy molecule is detected at a slow detection time.

As a matrix to be mixed with the sample, 2,5-dihydroxybenzoic acid (DHB), α-cyano-hydroxycinnamic acid (CHCA) and 3,5-dimethoxy-4-hydroxycinnamic acid (SA: Optimize different matrices for each characteristic.

For example, DHB and CHCA have been reported in the analysis of the PHMG. (Int. J. Mass Spectrom. 2013, 356, 1; BuLL. Korean Chem. Soc., 2013, 34, 1708)

In general, MALDI-TOF analysis for polymers is mainly based on qualitative analysis for acquiring information on repeating units (monomers), terminal groups, average molecular weight, and dispersion degree of the polymer. On the other hand, quantitative analysis of polymeric MALDI-TOF is not widely used due to low sample-to-sample, spot-to-spot and shot-to-shot reproducibility of MALDI- In particular, it is known that samples / matrices exhibit such a low reproducibility by producing very heterogeneous crystal structures.

However, recently, studies have been reported that quantitative analysis is obtained by obtaining a mass spectrometric spectrum including an internal standard material in MALDI-TOF measurement.

On the other hand, the unevenness of the sample / matrix crystal structure can be overcome by utilizing an ionic liquid matrix (ILM) (Anal. Chem. 2001, 73, 3679; , 2938)

The method for analyzing the oligomer content of a guanidine polymer in a household article according to the present invention comprises the steps of: a) extracting a sample solution containing an oligomer of a guanidine polymer from a household article; b) A sample for the internal reference material containing the ¹³ C-labeled guanidine polymer is mixed into the extracted sample solution, and the sample is prepared by changing the relative concentration of the sample solution and the composition for the internal standard material. Obtaining a MALDI-TOF (matrix-assisted Laser desorption / ionization time-of-flight) mass spectrometry spectrum; And c) quantitatively analyzing the oligomer content of the guanidine polymer in the sample solution by obtaining a calibration curve using each of the mass spectrometry spectra.

Here, the household article may be any one selected from a wet tissue, a contact lens cleaning liquid, a make-up remover, a hand cleaning liquid, a bactericide, a deodorant, and a fragrance, but is not limited thereto.

When quantitative analysis of a guanidine oligomer such as PHMG, PHMB, or PGH oligomer contained in the daily necessities is highly likely to exist as a background material in addition to the analyte in the daily commodity, the PHMG oligomer, the PHMB oligomer and the like Only the guanidine oligomer to be analyzed binds to the filler to remove the impurities in the sample, and at the same time, a selective pretreatment process capable of selective separation is required.

For example, in the case of wet tissues, polyethylene glycol (PEG) is often contained in excess as a water-containing agent. In order to quantitatively analyze PHMG and PHMB, it is necessary to develop a pretreatment process for selectively removing only PEG Do.

To this end, the sample solution is sampled by using a solid phase extractor (SPE) as a sample (a-1) between the steps a) and b) in quantitatively analyzing the oligomer content of the guanidine- And a step of purifying the solution.

The Solid Phase Extractor (SPE) is one of effective purification methods having a simple and easy to use method in the sample pretreatment. It is a generic term referring to cartridges filled with various fillers according to the type of the cartridge. It more effectively reduces the complicated sample preprocessing process.

The solid phase extraction cartridge can be used to separate only the analyte of interest from the interfering substances in the sample containing the analyte, and the respective substances can be selectively eluted by using different solvents.

In addition, since the filling agent filled in the cartridge is hydrophilic or hydrophobic, it can be divided into a Normanl phase cartridge and a reverse phase cartridge. In the normal phase cartridge, the filling material is made of a hydrophilic material It can be used for refining and extracting hydrophilic analytes such as glycans mainly by silica, and reverse phase cartridges can be used for purification and packing It is filled with a hydrophobic substance. C-18, in which 18 carbon atoms are connected in a chain form, and C-8, in which 8 carbon atoms are connected in a chain form, can be typically used. and can be used to purify and extract hydrophobic analytes such as proteins.

Therefore, the most important factor in the purification and extraction of analytes is to select cartridges filled with a suitable filler among the various solid phase extraction cartridges in consideration of the physical and chemical properties of the materials to be analyzed and the purpose of analysis. can do.

In the present invention, a solid phase extraction (SPE) purification method is used for separating and analyzing PHMG and / or PHMB present in daily necessities, and a mixed mode cation exchange , MCX) cartridge, it is possible to selectively extract only PHMG and PHMB components effectively by removing other background materials more easily.

In the present invention, by using the mixed mode cation exchange (MCX) cartridge, quantitative analysis can be facilitated by more effectively separating a guanidine oligomer having a guanidine group in a monomer such as PHMG, PHMB and PGH.

The mixed-mode cation exchange cartridge is composed of a filler in the form of a mixture of reversed phase / weak cation exchange beads. The filler component may be composed of a polymeric sorbent. For example, two monomeric lipophilic ) Of divinylbenzene and hydrophilic N-vinylpyrrolidone are blended in a specific ratio, and a polymer bead in which a polymer and a sulfonic acid are mixed is filled in a cartridge.

The packing agent packed in the cartridge contains a guanidine group NH ₂ ⁺ cationized to SO ₃ ^- of the packing material, NH ₃ ⁺ , and other impurities in the washing process during the pretreatment process are removed together with the washing solution to enable selective separation.

Particularly, since polyethylene glycol oligomers are contained in excessive amounts in wet tissues and the like, it is very important in quantitative analysis that these background substances are selectively removed. In the purification stage using the solid-phase extraction cartridge, the equilibrium state is created by activation of the cartridge and pre-wet process.

Methanol (MeOH) is preferably used as an activating solvent for the cartridge, 0.1% HCl / MeOH as a pre-wet process solvent, and 2 M HCl / MeOH as a solvent for final elution.

In the present invention, the mass spectrometry spectrum in step b) can be obtained by mixing a sample obtained by changing the relative concentration and an ionic liquid and using an ionic liquid matrix.

The ionic liquid matrix is a mixture of anionic and basic organic cationic materials used in conventional MALDI-TOF analysis, and generally a compound having a melting point of 100 DEG C or less can be used.

The most commonly used anionic materials are α-cyano-hydroxycinnamate (4-CHCA) and 2,5-dihydroxybenzoate (2,5-DHBA). Tributylamine, pyridine, 1-methylimidazole . An ionic liquid matrix made by mixing 1: 1 of these materials forms a very uniform thin film, and generally has a low vapor pressure.

On the other hand, when the internal standard material is used, the reliability of quantitative analysis using a mass spectrometer can be improved. In the case of MALDI-TOF, internal standards were also used for quantitative analysis. However, in these cases, internal standards were used only for monodisperse biomaterials such as peptides and carbohydrates. In the case of polymers or oligomers, , There is no example of quantitative analysis using an internal standard material. In particular, it is a technical feature of the present invention to use an internal standard material containing isotopes as in the present invention.

As an internal standard substance in the mass spectrometry according to the present invention, an internal standard substance substituted with a stable isotope may be used.

That is, when an internal reference material labeled with an isotope such as ¹³ C or ¹⁵ N is used for the sample to be analyzed, the internal standard material has almost the same chemical and physical properties as the analytical sample, It has the advantage of showing a slight difference in mass with the analytical sample, and has the advantage of mass analysis. It is essential to use an internal reference material labeled with an isotope in the quantitative analysis and it is necessary to use an internal reference material in which the oligomer in the guanidine polymer in the present invention can be reproduced well There are advantages.

The PHMG oligomers are classified into the following isomers according to the type of the repeating unit constituting the polymer, and they are classified as A-, B-, C-, D-, E-type PHMG oligomers.

In FIG. 1, a spectrum obtained by using a MALDI-TOF mass spectrometer is shown as a PHMG oligomer isomer. The A-, B-, C-, D- and E-type PHMG oligomers have a mass difference of 141 Da with oligomers of the following sizes and have the following masses depending on the number of repeating units.

At this time, one proton is attached to (M + H) ⁺ form in MALDI-TOF, and Table 1 below shows the mass including this proton.

Further, PHMB oligomers are classified into the following isomers according to the type of terminal group constituting the polymer, and they are represented by A-, B-, C-, D-, E-, F-, G-, H-, I-, J-, K-, L-, and M-type PHMB oligomers.

In FIG. 2, the spectrum obtained by using the MALDI-TOF mass spectrometer of the PHMB oligomer isomer is shown. The A-type to M-type PHMB oligomers have a mass difference of 185 Da from the oligomers of the next repeating unit size, and have the following masses depending on the number of the repeating units.

At this time, one proton is attached to (M + H) ⁺ form in MALDI-TOF, and Table 2 below shows the mass including this proton.

Also, the PGH oligomers are classified into the following isomers according to the type of terminal group constituting the polymer, and are classified into A-, B-, C-, and D-type PGH oligomers, respectively.

The A-, B-, C- and D-type PGH oligomers have a mass difference of 173 Da with oligomers of the following sizes and have the following masses according to the number of repeats:

At this time, one proton is attached to (M + H) ⁺ form in MALDI-TOF, and Table 3 below shows mass including this proton.

In the present invention, the method of analyzing the oligomer content of a guanidine polymer in a household article using the MALDI-TOF (Matrix-assisted Laser desorption / ionization time-of-flight) A) extracting a sample solution containing an oligomer of a polymeric polymer from the object to be detected; b) preparing a sample by varying the relative concentration of the sample solution and the composition for the internal standard material by mixing the extracted sample solution with a composition for internal standard labeled with a radioisotope, and preparing a sample from each MALDI-TOF -assisted Laser Desorption / Ionization Time-of-Flight) mass spectrometry spectrum; And c) obtaining a calibration curve using each of the mass spectrometry spectra, thereby quantitatively analyzing the oligomer content of the macromolecular polymer in the sample solution, and analyzing the oligomer content of the macromolecular polymer contained in the detection object Method can be provided.

In the present invention, the internal reference material is prepared using the compound labeled with ¹³ C in the present invention. However, in the present invention, the internal standard substance is prepared by adding ¹⁵ N Can be used even if a guanidine-based compound is used.

Also, the mass spectrometry spectrum in the step b) can be obtained by mixing an ionic liquid with a sample obtained by changing the relative concentration and using an ionic liquid mattress as described above.

It will be understood by those skilled in the art that quantitative analysis of the oligomer content of the guanidine based polymer will be apparent to those skilled in the art, and the detailed description will be fully understood from the foregoing description.

In the following description, MALDI-TOF mass spectrometry for the analysis of PHMG oligomers and PHMB oligomers present in household products is described as an exemplary embodiment of the method for analyzing the oligomer content of guanidine polymer. From this, PHMG and PHMB Analysis of the content of oligomers in similar guanidine based polymers would be equivocal to those of ordinary skill in the art.

≪ Preparation Example 1: Synthesis of ¹³ C-PHMG >

The synthesis of ¹³ C-PHMG is as shown in the following reaction formula.

First, hexamethylene diamine (hexamethylenediamine, 50.0 mg, 0.518 mmol ) and ¹³ C- guanidine hydrochloride 5 ml of cone ^{(13 C-guanidine hydrochloride, 60.2} mg, 0.518 mmol) in the same molar amount (cone) - into the vial open screw Close with cap (open screw cap) and septum.

The vial is heated at 100 < ⁰ > C with stirring using a stirring magnet. After heating for 1 hour in this state, the temperature is increased to 170 ^° C and the mixture is further heated for 6 hours.

Keep the needle in the septum so that ammonia can escape during heating. During the reaction, the reaction mixture becomes a yellowish sticky liquid, and ¹³ C-PHMG is obtained as a solid (80 mg, 79%) when cooled to room temperature. ¹ H NMR (400 MHz, (CD ₃ ) ₂ SO) 8.34 - 6.79 (brm, 7H) , 3.33 (s, H 2 O), 3.10 (m, 4H), 2.50 (m, DMSO), 1.45 (m, 4H), 1.31 (m, 4H); ¹³ C NMR (400 MHz, (CD ₃ ) ₂ SO) δ 157.04, 155.83, 40.45 (DMSO), 28.22, 25.50.

3 and 5 in the ¹³ C-PHMG ¹ H-NMR of the showed a spectrum obtained from a ¹³ C-NMR, FIG. 7, the composite ¹³ C-PHMG is obtained by using a MALDI-TOF mass spectrometry spectrum And the mass values of the synthesized ¹³ C-PHMG isomeric oligomers are shown in Table 4 below.

≪ Preparation Example 2: Synthesis of ¹³ C-PHMB >

¹³ The synthesis of C-PHMB is shown in the following reaction formula.

1) Synthesis of 1, 6- ¹³ C ₂ -hexamethylenediamine dihydrochloride

First, 1, 6- ¹³ C ₂ - hexamethylene diamine _{^{(1, 6- 13 C 2 -hexamethylenediamine}} , 55 mg, 0.465 mmol) were placed in a 1 dram (4 mL) a vial and dissolved in 1 mL methanol. Trimethylsilyl chloride (126 mg, 1.16 mmol) was added dropwise thereto. The solution was stirred at room temperature for 30 minutes, and the solvent and volatile materials were all removed using a vacuum rotary evaporator. The product thus obtained was used without further purification. (83 mg, 93%): 1 H NMR (400 MHz, (CD 3) 2 SO) δ 8.22 - 7.71 (brs, 6H), 3.33 (s, H 2 O), 2.75 (m, 2H), 2.50 ( m, DMSO), 1.54 (m, 2H), 1.31 (m, 2H); ¹³ C NMR (400 MHz, (CD ₃ ) ₂ SO)? 40.45 (DMSO), 39.29, 26.95, 25.74.

_{^{2) 1, 6- 13 C 2}} - Synthesis of bis FIG guanidyl-hexane _{^{(1, 6- 13 C 2 -Bisguanidohexane}} )

1, 6- ¹³ C ₂ - hexamethylene diamine dihydrochloride _{^{(1, 6- 13 C 2 -hexamethylenediamine}} dihydrochloride, 40 mg, 0.21 mmol) and sodium amide is moved Ian (sodium dicyanamide, 37 mg, 0.42 mmol) to 1 < / RTI > dram vials. This mixture was dissolved in 1 mL of n-butanol and heated to 135 DEG C for 8 hours. After the reaction was completed, the mixture was allowed to cool at room temperature and the solid began to precipitate. The solid was filtered, washed with n-butanol, and the solvent of the filtrate was removed with a vacuum rotary evaporator. The solid thus obtained was recrystallized from water and filtered to obtain a white solid. (53 mg, 100%): ¹ H NMR (400 MHz, (CD ₃ ) ₂ SO)? 7.99 - 6.21 (brm, 6H), 3.33 (s, H ₂ O), 3.01 m, DMSO), 1.39 (m, 2H), 1.23 (m, 2H); ¹³ C NMR (400 MHz, (CD ₃ ) ₂ SO) δ161.57, 118.80, 41.08, 40.45 (DMSO), 29.29, 26.30.

3) Synthesis of ¹³ C-PHMB ( ¹³ C-polyhexamethylene biguanide)

1 of the same number of equivalents, 6- ¹³ C ₂ - bis gu cyano no dino-hexane _{^{(1, 6- 13 C 2 -biscyanoguanidinohexane}} , 30 mg, 0.157 mmol) and 1, 6- ¹³ C ₂ - hexamethylene diamine die Hydrochloride (1, 6- ¹³ C ₂ -hexamethylenediamine dihydrochloride, 40 mg, 0.157 mg) was added to a 5 mL cone vial and dissolved in 3 mL of distilled water. The solution was stirred for 30 minutes while being heated to 120 DEG C to completely blow off water. Thereafter, the temperature was raised to 160 DEG C and the reaction was further continued for 2 hours. After the reaction was terminated, the product was reduced in pressure to remove all of the water, thereby obtaining a yellow solid product without further purification. (64 mg, 91%): ¹ H NMR (400 MHz, (CD ₃ ) ₂ SO)? 7.88-6.49 (brm, 7H), 3.33 (s, H ₂ O), 3.08 s, DMSO), 1.44 (m, 4 H), 1.29 (m, 4 H); ¹³ C NMR (400 MHz, (CD ₃ ) ₂ SO) δ161.71, 118.86, 41.13, 40.45 (DMSO), 38.97, 29.10, 25.7. Overall Yield = 85%

4 and 6, the ¹³ C-PHMB ¹ H-NMR of the showed a spectrum obtained from a ¹³ C-NMR, FIG. 8, a synthetic ¹³ C-PHMB obtained using MALDI-TOF mass spectrometry spectrum And the mass values of the synthesized ¹³ C-PHMG isomeric oligomers are shown in Table 5 below.

≪ Example 1: Preparation of PHMG quantitative assay line using SPE purification method >

1) Purification is carried out by collecting the solution obtained from the household products and putting the ¹³ C-labeled PHMG together with the internal standard material and using the solid phase extraction cartridge. A sample preprocessing process is required to extract the sample from the cartridge. The aliquot is diluted with a methanol solution at a ratio of 1: 3 (v: v), and a small amount of 0.1% HCl / MeOH solution is added to acidify the sample solution.

2) Loading the sample into the cartridge in equilibrium with the activation and pre-wet process of the cartridge as described above. Then, after several washing steps using 0.1% HCl / MeOH solution, the analytes are eluted by eluting the eluting solvent 2M HCl / MeOH in the filler.

3) The solid phase extracted PHMG component and ¹³ C PHMG internal standard substance in the above step are prepared in a solid state by vacuum rotation-drying. To quantitatively analyze the samples for reproducibility and reliable MALDI-TOF, samples are prepared for MALDI-TOF mass spectrometry using a 1-mehtylimidazole /? -Cyano-hydroxycinnamate ionic liquid matrix of Formula 1 below.

[Chemical Formula 1]

The 1-mehtylimidazole / α-cyano-hydroxycinnamate ionic liquid matrix was prepared by dissolving 0.2 mol α-Cyano-4-hydroxycinnamic acid in methanol and adding 0.2 mol 1-methylimine Prepare by adding 1-Methyl imidazole.

Mix well for 1 minute with a Vortex mixer and leave at room temperature for 5 to 10 minutes to allow the reaction to proceed. Fill the solvent with an evaporator and weigh again to dilute with methanol to a concentration of 70 mg / mL. After the completely dried analyte is dissolved again in water, the analyte is mixed with the matrix at a ratio of 1: 1000 and loaded onto a MALDI sample plate (eg, anchor chip sample plate (MTP AnchorChip 384 BC, Part.no 8280790, Bruker) And dried thoroughly before analysis using a MALDI-TOF mass spectrometer.

The MALDI-TOF mass spectrometry spectrum is then obtained by varying the relative concentrations of the PHMG and ¹³ C-labeled PHMG internal standard material to form a calibration curve.

<Summary of calibration straight line for PHMG quantitative analysis>

1) Wet tissue solution (without ¹² C PHMG) 800 μL + ¹² C Mix 100 μL of PHMG standard solution and 100 μL (1000 ppm) of internal standard (PHMG labeled with ¹³ C).

( ¹² C PHMG concentration: 10000, 5000, 1000, 500, 100, 50, 10 ppm)

① Add 3 μL of 0.1% HCl / MeOH solution to acidify the sample.

② Dilute the sample with 3 mL of methanol.

2) Condition and equilibrate the solid phase extraction cartridge.

(Used cartridge: Waters' Oasis MCX (Mixed-mode Cation Exchange) cartridge)

① Conditioning: Flow the cartridge 3 times with 1 mL of methanol.

② Equilibrium: Flow the cartridge 3 times with 1 mL of 0.1% HCl / MeOH solution.

3) Load the sample (4 mL) into the cartridge.

① Drain 0.1% HCl / MeOH solution 2 ~ 3 times in 1 mL increments to remove impurities.

② Dilute 1 mL of 2M HCl / MeOH solution to elute the sample.

4) The eluate is dried using a vacuum rotary evaporator.

5) Dissolve the dried sample again in 1 mL of distilled water.

6) Mix well with 3 μL of sample and 3 μL of ILM-CHCA (ionic liquid matrix), and load 5 μl of 1 μL into the MALDI sample plate. Finally, draw a black straight line.

(X-axis is the logarithmic logarithm of ¹² C PHMG concentration, Y-axis is the logarithm of the ratio of ¹² C PHMG and internal standard ( ¹³ C-labeled PHMG)

Here, in the case of using MALDI-TOF mass spectrometer, 355 nm Nd: YAG laser light source was used for analysis, and N ₂ laser may be used. The laser intensity was set to a value of "Reflectron voltage" of + 2,164 V, "Laser percentage" to prevent fragmentation and fragmentation of the guanidine based polymer and to reduce the disproportion of distribution caused by the relatively low desorption effect of relatively large PHMG molecules "To 50% and" Suppress to 430 Da ".

Table 6 below shows experimental values of the calibration curve for ¹² C PHMG quantitative analysis.

FIG. 9 is a MALDI-TOF spectrum according to ¹² C PHMG concentration based on Table 6. FIG. According to Figure 9, a MCX cartridge and mixed with pre-treatment of the solid phase extraction process and with the overall of ¹³ C ¹² C PHMG PHMG as an internal standard at various concentrations, obtained by mass analysis using MALDI-TOF spectra.

The concentration of ¹² C PHMG from the top of FIG. 9 is 1000 ppm, 500 ppm, 300 ppm, 100 ppm, 50 ppm, 10 ppm, 5 ppm, 1 ppm and the concentration of the internal standard is fixed at 100 ppm. From the top down, the concentration of ¹² C PHMG decreased from 1000 ppm to 1 ppm. As a result, the spectral peaks of ¹³ C labeled PHMGs gradually increased, and the relative intensities were reversed. (A ₃ : A type ¹² C PHMG, A ₃ ^* : A type ¹³ C PHMG, C ₃ : C type ¹² C PHMG, C ₃ ^* : C type ¹³ C PHMG)

11 shows the results obtained by using the black linear standard solution " ¹² C-PHMG-free wet tissue solution + ¹² C-PHMG standard solution + internal standard substance ( ¹³ C-PHMG) solution" ^12C -PHMG linear calibration curve based on MALDI-TOF spectral data obtained for each ¹² C-PHMG concentration in FIG. The y-axis values are obtained by dividing the sum of the areas of the ion peaks corresponding to ¹² C-PHMG in the mass spectrometry spectrum by the sum of the areas of the ion peaks of the internal standard ( ¹³ C-PHMG) And the values on the x-axis are the logarithm of the ¹² C-PHMG concentration used in each measurement experiment.

The black line is represented by y = ax + b, where y is the peak area ratio of the selected ions of the reference material and the internal standard, x is the concentration ratio of the reference material and internal standard, a is the slope of the black line, It means a piece of black straight line. In this experiment, a and b values of the straight line are determined.

<Example 2: Preparation of PHMB quantitative calibration line using SPE purification method>

In order to quantify the PHMB in dairy products, but the same as the process in Example 1 was collected using a ¹³ C-labeled PHMB in place of ¹³ C-labeled PHMG as an internal standard.

Subsequently, the MALDI-TOF mass spectrometry spectrum is obtained by varying the relative concentrations of the PHMB and ¹³ C labeled PHMB internal standard materials, and a calibration curve is created.

<Summary of Calibration Straight Line for PHMB Quantitative Analysis>

1) Wet tissue solution (without ¹² C PHMB) 800 μL + ¹² C PHMB Standard solution 100 μL + 100 μL internal standard ( ¹³ C labeled PHMB) are mixed.

( ¹² C PHMB concentration: 10000, 5000, 1000, 500, 100, 50, 10 ppm)

① Add 3 μL of 0.1% HCl / MeOH solution to acidify the sample.

② Dilute the sample with 3 mL of methanol.

2) Condition and equilibrate the solid phase extraction cartridge.

(Used cartridge: Waters' Oasis MCX (Mixed-mode Cation Exchange) cartridge)

① Conditioning: Flow the cartridge 3 times with 1 mL of methanol.

② Equilibrium: Flow the cartridge 3 times with 1 mL of 0.1% HCl / MeOH solution.

3) Load the sample (4 mL) into the cartridge.

① Drain 0.1% HCl / MeOH solution 2 ~ 3 times in 1 mL increments to remove impurities.

② Dilute 1 mL of 2 M HCl / MeOH solution to elute the sample.

4) The eluate is dried using a vacuum rotary evaporator

5) Dissolve the dried sample again in 1 mL of distilled water.

6) Mix well with 3 μL of sample, 3 μL of ILM-CHCA (ionic liquid matrix), and load 5 μl of 1 μL into MALDI sample plate. Finally, draw a black straight line.

(The logarithm of the logarithmic value of the ¹² C PHMB concentration on the X axis and the logarithm of the ratio of the ¹² C PHMB and the internal standard ( ¹³ C labeled PHMB) on the Y axis)

For MALDI-TOF mass spectrometry, 355 nm Nd: YAG laser source was used and N ₂ laser may be used. The laser intensity is set to a value of "Reflectron voltage" of + 2,164 V, "Laser percentage" to prevent fragmentation and fragmentation of the guanidine based polymer and to reduce the disproportionation of distribution caused by the relatively low desorption effect of relatively large PHMB molecules "To 50% and" Suppress to 310 Da ".

Table 7 below shows the experimental values of the calibration curve for ¹² C PHMB quantitative analysis.

10 is a MALDI-TOF spectrum according to ¹² C PHMB concentration based on Table 7. Referring to FIG. 10, the mass spectra obtained by mixing various concentrations of ¹² C PHMB and ¹³ C PHMB, which are an internal standard substance, in an MCX cartridge solid phase extraction treatment and various preprocessing, followed by analysis through MALDI-TOF.

From the top of FIG. 10, the concentration of ¹² C PHMB is 1000 ppm, 500 ppm, 300 ppm, 100 ppm, 50 ppm, 10 ppm, 5 ppm, 1 ppm, and the concentration of the internal standard is fixed at 100 ppm. From the top down, the concentration of ¹² C PHMB decreased from 1000 ppm to 1 ppm. As a result, the spectral peaks of the PHMB labeled with ¹³ C, which is an internal standard, were gradually increased, and the relative intensities were reversed. (A _1: A Type _{^{12 C PHMB, A 1 *:}} A Type ¹³ C PHMB, B _1: B Type _{^{12 C PHMB, B 1 *:}} B Type ¹³ C PHMB, C _1: C type ¹² C PHMB, C ₁ ^* : C type ¹³ C PHMB, F ₁ : F type ¹² C PHMB, F ₁ ^* : F type ¹³ C PHMB, H ₁ : H type ¹² C PHMB, H ₁ ^* : H type ¹³ C PHMB)

12 shows a black straight line obtained according to Example 2 using a black linear standard solution " ¹² C-PHMB-free wet tissue solution + ¹² C-PHMB standard solution + internal standard material ( ¹³ C-PHMB) solution" And ¹² C-PHMB linear calibration curve based on MALDI-TOF spectral data obtained for each ¹² C-PHMB concentration in FIG. ¹² . The y-axis values were obtained by dividing the sum of the areas of the ion peaks corresponding to ¹² C-PHMB in the mass spectrometry spectrum by the sum of the areas of the ion peaks of the internal standard ( ¹³ C-PHMB) Value. The values on the x-axis are the logarithm of the ¹² C-PHMB concentration used in each measurement.

&Lt; Example 3: Selection of cartridge >

In analyzing a guanidine oligomer such as PHMG or PHMB present in daily necessities, it is necessary to select an appropriate cartridge to selectively remove PEG, which is the substance exhibiting the largest background noise. In the present invention, a cartridge suitable for quantitative analysis of guanidine oligomers For selection, a mixture of PHMG and PEG, which is one of the guanidine oligomers, was prepared. Four cartridges among four types of solid phase extraction (SPE) cartridges used for general use were selected and purified. Then, a MALDI-TOF mass spectrometer Respectively.

13 is a mass spectrometry spectrum obtained using a MALDI-TOF mass spectrometer of a solid-phase extraction cartridge.

13 (a) is a spectrum obtained by mixing a suitable mixture of PHMG and PEG as a reference cartridge using a MALDI-TOF mass spectrometer. Through the above analysis, the oligomer distribution in which PHMG and PEG appear can be confirmed.

13 (b), PHMG was purified using a C-18 cartridge widely used in solid-phase extraction cartridges. The filler of the C-18 cartridge is represented by the following chemical formula 2, and the solvent used is 0.1% TFA / H2O in the washing process and TA50 (in terms of 10 mL, Acetonitrile: 5 mL, H ₂ O: : 400 μL) was used.

(2)

13 (c) is a Titanium Dioxide cartridge. The structure of the filler is shown in the following chemical formula 3. The solvents used were 20 mg / mL DHB (2,5-Dihydroxybenzoic acid) / TA10 and TA10 (1 mL, Acetonitrile: 1 mL, H ₂ O: 8.6 mL, 2.5% TFA: 400 μL) In the procedure, TA80 (1 mL, Acetonintrile: 8 mL, H ₂ O: 1.6 mL, 2.5% TFA: 400 μL) was used.

(3)

13 (d) is a Porous Graphite Carbon Black cartridge. The structure of the filler is shown in Formula 4 below. The solvent used was H ₂ O in the washing process and TA50 (10 mL, Acetonitrile: 5 mL, H ₂ O: 4.6 mL, 2.5% TFA: 400 μL) in the elution procedure.

[Chemical Formula 4]

13 (e) is a Mixed-mode Cation Exchange cartridge. The structure of the filler is shown in the following Chemical Formula 5. It has a pK _a <1, wettability of the filler, and can be used stably in a wide range of pH range from 0 to 14, which is used in the form of a mixture of reversed phase / weak cation exchange beads. The basic molecules can be selectively separated and are stable to organic solvents. The solvent used was 0.1% HCl / MeOH in the washing process and 2 M HCl / MeOH in the eluting process.

[Chemical Formula 5]

According to Examples 1 and 2, an improved effect can be exhibited by carrying out an impurity removal process to qualitatively and quantitatively analyze a guanidine oligomer present in household goods using a MALDI-TOF mass spectrometer. Mixed-mode cation exchange cartridges (MCX) among the solid-phase extraction cartridges can be optimized to remove the background noise and selectively isolate only the PHMG and PHMB.

<Example 4: PHMG analysis of wipes manufactured in Korea using solid-phase extraction cartridge>

1) Wet the wipes manufactured in Korea using a plastic syringe, collect 900 μL of wet tissue solution, and mix well with 100 μL of ¹³ C-labeled PHMG as an internal standard.

2) Add 3 mL of methanol, add 3 μL of 0.1% HCl / MeOH to acidify the solution, and mix again for 1 minute using a Vortex mixer.

3) Activate and pre-wet the MCX cartridge for solid phase extraction. Activation of the cartridges is carried out at 3 times the volume of the cartridge, 3 ml of methanol if a 1 cc capacity cartridge is used, and 3 times the volume of the cartridge with a 0.1% HCl / MeOH pre-wet process. In case of using, 3 mL of methanol is poured 3 times in 1 mL, and the condition of the cartridge is maintained.

4) Load the sample onto the cartridge. When loading the first cartridge, the inlet of the cartridge is clogged and the solvent does not penetrate well. Therefore, it is recommended that the cartridge be pushed slowly using nitrogen or air pressure. All samples are loaded and then washed (0.1% HCl / MeOH). In the case of the washing process, the number of times may be reduced or increased depending on the amount of impurities.

5) At the end of the washing process, elute the analytical material by eluting the 2 M HCl / MeOH solvent in the cartridge, dry the solvent completely by using a vacuum rotary evaporator, and obtain a sample in a solid state.

6) Dissolve completely dried sample in 1 mL of water. When the sample is ready, mix 1 μL of the analyte and 1 μL of the matrix ILM-CHCA (ionic liquid matrix), and load 1 μL into the anchor chip MALDI sample plate. The sample plate is placed in a desiccator and the solvent is completely dried and analyzed.

14 shows a mass spectrometry spectrum obtained using the MALDI-TOF mass spectrometer of Example 4. As a result of spectral analysis of the MALDI-TOF mass spectrometer, the content of PHMG in the wet tissue produced in Korea was below the detection limit, Is the ¹³ C-PHMG, the internal standard used.

<Example 5: PHMG analysis of wipes manufactured in China using solid-phase extraction cartridge>

1) Squeeze the wipes containing PHMG (Country of Origin: China) with Norm Jet plastic syringe and obtain 900 μL of the saline solution. Add 100 μL of ¹³ C-labeled PHMG 1000 ppm as an internal standard give.

2) Dilute 3 mL of methanol in 1 mL of sample solution, add 3 μL of 0.1% HCl / MeOH to acidify. Mix the sample thoroughly with a vortex mixer for 1 minute.

3) Activate and pre-wet the MCX cartridge (Mixed-mode Cation Exchange) to purify PHMG using a solid-phase extraction cartridge. Activate the cartridge by three times the volume of the cartridge, and if using a 1 cc capacity cartridge, flow 3 mL of methanol. Apply 3 mL of 1 mL each time to maintain the condition of the cartridge. The pre-wet procedure is to equilibrate the condition with 3 times the volume of the cartridge with 0.1% HCl / MeOH, 3 mL of 0.1% HCl / MeOH 3 mL when using 1 cc capacity cartridges.

4) After loading the pretreated sample in the equilibrium cartridge, the washing process is performed to remove impurities other than the substance to be analyzed. The washing process is performed by flowing 1 mL of the 0.1% HCl / MeOH solution previously prepared. It is preferable to wash 0.1% HCl / MeOH solution three times as much as the volume of the cartridge, and if there are many impurities, it is preferable to thoroughly wash by adjusting the number of times of washing.

5) After the washing process, the analyte is separated from the cartridge filler and eluted with the eluent. The eluate is eluted with 2 M HCl solution 2 mL in 1 mL each time. The solvent is then completely dried using a vacuum rotary evaporator to obtain a solid sample.

6) Dissolve completely dried sample in 1 mL of water. 1 μL of the prepared sample and 1 μL of matrix ILM-CHCA (ionic liquid matrix) are mixed well and analyzed by loading 1 spot each 1 μL onto anchor chip MALDI plate. Use a MALDI-TOF mass spectrometer to analyze the dried sample on the plate after placing the sample plate in a desiccator to completely dry the solvent.

15 is a mass spectrometry spectrum obtained using the MALDI-TOF mass spectrometer of Example 5. Fig. 15 (a) is a mass spectrometry spectrum obtained by using a MALDI-TOF mass spectrometer according to Example 5, and (b) shows the peak of A ₃ type and C ₃ type It is the enlarged spectrum. The pattern on the side of the labeled peak indicated the peak of ¹³ C PHMG, the internal standard used.

In this spectrum, it can be seen that the various types of PHMG structures and monomers differ from each other by 141 types, and the polymer distribution can be confirmed by types A, B and C.

The mass value of the spectrum of FIG. 15 (a) is shown in Table 8, and the PHMG content analysis statistical data present in the wet tissue (country of manufacture: China) are shown in Table 9.

In order to obtain a calibration curve, two Chinese wipes were squeezed out and well mixed with an internal standard material, and each was pretreated with a cartridge. The pretreated analytes were diluted again and then loaded with 1 spots each with 1 μl. Also, 10 shots of 1000 shots were combined and a spectrum was obtained using a MALDI-TOF mass spectrometer.

Obtain two total spectra and obtain relative ratios through Σ (sum of all PHMG ion peaks) / Σ (sum of all ¹³ C isotope-substituted PHMG ion peaks). The obtained y value is substituted into the formula obtained through the previously drawn black straight line. Since x = log [PHMG], the logarithm value of the PHMG content in the wet tissue can be determined as x value by calculation. Therefore, the content of PHMG present in the wet tissue can be obtained by [PHMG] = 10 ^-x .

<Example 6: PHMB analysis of wipes manufactured in China using solid-phase extraction cartridge>

The analysis was carried out in the same manner as in Example 4 except that the analytes were analyzed using a wet tissue manufactured in China instead of the wipes produced in Korea and the PHMB oligomer was analyzed in place of the PHMG oligomer.

FIG. 16 shows a mass spectrometry spectrum obtained using the MALDI-TOF mass spectrometer of Example 6, and a portion marked with an asterisk (*) next to the labeled peak is a ¹³ C-labeled PHMB used as an internal standard.

16 are shown in Table 10, and the PHMB content analysis statistical data present in the wet tissue are shown in Table 11. [

In order to obtain a calibration curve, the wipes (country of origin: China) were plucked and kneaded well, mixed well with the internal standard material, and each was pretreated with a cartridge. The pretreated analytes were diluted again and then loaded with 1 spots each with 1 μl. Also, 10 shots of 1000 shots were combined and a spectrum was obtained using a MALDI-TOF mass spectrometer. Obtain two total spectra and obtain relative ratios through Σ (sum of all PHMB ion peaks) / Σ (sum of all ¹³ C isotope-substituted PHMB ion peaks) and then calculate the y value by taking the log value. The obtained y value is substituted into the formula obtained through the previously drawn black straight line. Since x = log [PHMB], the logarithm of the PHMB content in the wet tissue can be obtained as x value by calculation. Therefore, the PHMB content in the wet tissue can be obtained by [PHMB] = 10 ^-x .

<Example 7: PHMG analysis in a contact lens cleaning liquid using a solid-phase extraction cartridge>

1) In case of contact lens cleaner, since it exists in liquid state at room temperature, 900 μL is obtained from the liquid phase itself, and the internal standard substance, ¹³ C labeled PHMG 1000 ppm and 100 μL is mixed well.

2) Dilute 3 mL of methanol in 1 mL of sample solution, add 3 μL of 0.1% HCl / MeOH to acidify. Mix the sample thoroughly with a vortex mixer for 1 minute.

3) Activate and pre-wet the MCX cartridge (Mixed-mode Cation Exchange) to purify PHMG using a solid-phase extraction cartridge. Activate the cartridge by three times the volume of the cartridge, and if using a 1 cc capacity cartridge, flow 3 mL of methanol. Apply 3 mL of 1 mL each time to maintain the condition of the cartridge. The pre-wet procedure is to equilibrate the condition with 3 times the volume of the cartridge with 0.1% HCl / MeOH, 3 mL of 0.1% HCl / MeOH 3 mL when using 1 cc capacity cartridges.

4) After loading the pretreated sample in the equilibrium cartridge, the washing process is performed to remove impurities other than the substance to be analyzed. The washing process is performed by flowing 1 mL of the 0.1% HCl / MeOH solution previously prepared. It is preferable to wash 0.1% HCl / MeOH solution three times as much as the volume of the cartridge, and if there are many impurities, it is preferable to thoroughly wash by adjusting the number of times of washing.

5) After the washing process, the analyte is separated from the cartridge filler and eluted with the eluent. The eluate is eluted with 2 M HCl solution 2 mL in 1 mL each time. The solvent is then completely dried using a vacuum rotary evaporator to obtain a solid sample.

6) Dissolve completely dried sample in 1 mL of water. 1 μL of the prepared sample and 1 μL of matrix ILM-CHCA (ionic liquid matrix) are mixed well and analyzed by loading 1 spot each 1 μL onto anchor chip MALDI plate. The sample plate is placed in a desiccator, the solvent is completely dried, and then a MALDI-TOF mass spectrometer is used.

17 is a mass spectrometry spectrum obtained using the MALDI-TOF mass spectrometer of Example 7 according to the present invention. As a result of spectral analysis, PHMG peaks other than ¹³ C - labeled PHMG peaks were not confirmed. Therefore, PHMG content in the contact lens cleaning solution was found to be below the detection limit or not. The pattern on the side of the labeled peak indicates the peak of the internal reference material, ¹³ C labeled PHMG used.

<Example 8: PHMB analysis in a contact lens cleaning liquid using a solid-phase extraction cartridge>

The same procedure as in Example 7 was carried out to analyze PHMB oligomers instead of PHMG oligomers.

18 shows a mass spectrometry spectrum obtained using the MALDI-TOF mass spectrometer of Example 8. The portion marked with an asterisk (*) next to the labeled peak is a ¹³ C-labeled PHMB used as an internal standard material.

The spectral mass values in FIG. 18 are shown in Table 12 below, and PHMB content analysis statistical data present in the contact lens cleaning solution are shown in Table 13 below.

Further, to obtain a calibration straight line, the contact lens cleaning liquid was collected twice, mixed well with an internal standard material, and pretreated with a cartridge. The pretreated analytes were diluted again and then loaded with 1 spots each with 1 μl. Also, 10 shots of 1000 shots were combined and a spectrum was obtained using a MALDI-TOF mass spectrometer. Obtain two total spectra and obtain relative ratios through Σ (sum of all PHMB ion peaks) / Σ (sum of all ¹³ C isotope-substituted PHMB ion peaks) and then calculate the y value by taking the log value. The obtained y value is substituted into the formula obtained through the previously drawn black straight line. x = log [PHMB], so that the logarithm of the PHMB content in the contact lens cleaning liquid can be obtained as an x value by calculation. Therefore, the PHMB content in the contact lens cleaning liquid can be obtained by [PHMB] = 10 ^-x .

&Lt; Example 9: PHMG analysis in a make-up remover using a solid phase extraction cartridge >

The same procedure as in Example 7 was followed to analyze the sample, and the PHMG oligomer contained therein was quantitatively analyzed using the sample to be analyzed as a make-up remover.

19 is a mass spectrometry spectrum obtained using the MALDI-TOF mass spectrometer of Example 9. Fig. As a result of spectral analysis, PHMG peaks other than ¹³ C - labeled PHMG peaks were not confirmed. As a result, PHMG content in the make - up remover was found to be below the detection limit. Next to the labeled peaks here, the * pattern indicates the peak of the internal reference material, ¹³ C-labeled PHMG used.

&Lt; Example 10: PHMB analysis in a make-up remover using a solid phase extraction cartridge >

The same procedure as in Example 9 was carried out to analyze PHMB oligomers instead of PHMG oligomers.

20 shows the mass spectrometry spectrum obtained using the MALDI-TOF mass spectrometer of Example 10. The portion marked with an asterisk (*) next to the labeled peak is the ¹³ C-labeled PHMB used as the internal standard used.

In addition, the mass values of the spectrum of FIG. 20 are shown in the following Table 14, and the PHMB content analysis statistical data present in the make-up remover are shown in Table 15 below.

In order to obtain a calibration curve, the decoloring agent was collected twice, mixed well with an internal standard material, and pretreated with a cartridge. The pretreated analytes were diluted again and then loaded with 1 spots each with 1 μl. Also, 10 shots of 1000 shots were combined and a spectrum was obtained using a MALDI-TOF mass spectrometer. Obtain two total spectra and obtain relative ratios through Σ (sum of all PHMB ion peaks) / Σ (sum of all ¹³ C isotope-substituted PHMB ion peaks) and then calculate the y value by taking the log value. The obtained y value is substituted into the formula obtained through the previously drawn black straight line. Since x = log [PHMB], the logarithm of the PHMB content in the make-up remover can be calculated as x value. Therefore, the PHMB content in the make-up remover can be obtained by [PHMB] = 10 ^-x .

&Lt; Example 11: Qualitative analysis of PGH in mixture using SPE purification method >

1) Add 500 μL of the PGH standard solution with a concentration of 1000 ppm and 500 μL of the PEG standard solution with a concentration of 100 ppm, and mix well to make a mixture.

2) Dilute 3 mL of methanol in 1 mL of sample solution, add 3 μL of 0.1% HCl / MeOH to acidify. Mix the sample thoroughly with a vortex mixer for 1 minute.

3) Activate and pre-wet the MCX cartridge (Mixed-mode Cation Exchange) to purify PGH using a solid-phase extraction cartridge. Activate the cartridge by three times the volume of the cartridge, and if using a 1 cc capacity cartridge, flow 3 mL of methanol. Apply 3 mL of 1 mL each time to maintain the condition of the cartridge. The pre-wet procedure is to equilibrate the condition with 3 times the volume of the cartridge with 0.1% HCl / MeOH, 3 mL of 0.1% HCl / MeOH 3 mL when using 1 cc capacity cartridges.

4) After loading the pretreated sample in the equilibrium cartridge, the washing process is performed to remove impurities other than the substance to be analyzed. The washing process is performed by flowing 1 mL of the 0.1% HCl / MeOH solution previously prepared. It is preferable to wash 0.1% HCl / MeOH solution three times as much as the volume of the cartridge, and if there are many impurities, it is preferable to thoroughly wash by adjusting the number of times of washing.

5) After the washing process, the analyte is separated from the cartridge filler and eluted with the eluent. The eluate is eluted with 2 M HCl solution 2 mL in 1 mL each time. The solvent is then completely dried using a vacuum rotary evaporator to obtain a solid sample.

6) Dissolve completely dried sample in 1 mL of water. 1 μL of the prepared sample and 1 μL of matrix ILM-CHCA (ionic liquid matrix) are mixed well and analyzed by loading 1 spot each 1 μL onto anchor chip MALDI plate. The sample plate is placed in a desiccator, the solvent is completely dried, and then a MALDI-TOF mass spectrometer is used.

21 and 22 are mass spectrometry spectra obtained using the MALDI-TOF mass spectrometer of Example 11. More specifically, FIG. 21 shows the results of mass spectrometry of a mixture of PGH and PEG at a specific concentration, FIG. 22 is a MALDI-TOF mass spectrometry spectrum obtained without a pretreatment process of a mass spectrometric spectrometer using a mixed-mode cation exchange cartridge. to be.

In the spectrum of FIG. 21, it can be confirmed that PEG is analyzed like PGH. On the other hand, in the spectrum of FIG. 22, it can be seen that after pretreatment using the mixed mode cation exchange cartridge, have.

The spectral mass values in FIG. 22 are shown in Table 16 below.

According to Example 11, PGH has a guanidine group in a monomer such as PHMG and PHMB, and it was confirmed that PEG was removed through a pretreatment with an appropriate amount of PGH and PEG mixture through a mixed mode-cation exchange cartridge, and PGH It is possible to purify from other impurities through mixed mode-cation exchange cartridges in solid phase extraction cartridges such as PHMG, PHMB.

Claims (18)

A guanidine group or a guanidine group, wherein the carbon linked to the three nitrogen atoms in the guanidine group is substituted with ¹³ C and labeled guanidine group or a guanidine group. The method according to claim 1,
Wherein the guanidine polymer comprises a repeating unit of any one of the following structural formulas A to D:
[Structural Formula A]

[Structural formula B]

[Structural formula C]

[Structural formula D]

Provided that the repeating unit of each compound in Structural Formulas A to D is 1 to 1000. [ Guanidine group or a guanidine group, wherein an alkylene group having 1 to 12 carbon atoms is bonded to any one nitrogen atom in the guanidine group, or an alkylene group having 1 to 12 carbon atoms is bonded to any nitrogen atom in the bivalent group And at least one of the carbon atoms in the alkylene group is substituted with ¹³ C, and the labeled guanidine polymer is labeled. The method according to claim 1 or 3,
The guanidine-based polymer is an oligonucleotide labeled with ¹³ C - [2- ethoxyethyl guanidinium chloride (ethoxy 2-) (oligo- [2- (2 -ethoxy) -ethoxyethyl] -guanidinium chloride; PGH), 13 the labeled as C polyhexamethylene guanidine phosphate (Polyhexamethyleneguanidine phosphate) or ¹³ C as the polyhexamethylene guanidine hydrochloride (Polyhexamethyleneguanidine hydrochloride) or ^13. the polyhexamethylene biguanides hydrochloride (polyhexamethylene Biguanide hydrochloride) labeled with C or ¹³ labeled C Wherein the guanidine polymer is any one of polyhexamethylene biguanidinium phosphate labeled with a guanidine group. A ¹³ C-labeled polyhexamethylene guanidine (PHMG) polymer obtained by the reaction of the ¹³ C-labeled guanidine salt represented by the following structural formula A-1 and the hexamethylenediamine represented by the structural formula B-1.

Structural Formula A-1 Structural Formula B-1
X in the structural formula A-1 is a halogen or a monovalent anion. The following structural formulas A-2 to the structural formula A-4 or more of any of the ¹³ C is represented by a labeled guanidine salt and the structural formula B-1 obtained by the reaction of hexamethylene diamine, ¹³ C-labeled polyhexamethylene biguanides (PHMB) polymer .

Structure A-2

Structure A-3

Structural Formula A-4 Structural Formula B-1
Provided that X in the structural formulas A-2 to A-4 is a halogen or a monovalent anion. The following structural formula A-5 to A-6 one of the compound and, ¹³ C-labeled polyhexamethylene biguanides (PHMB) obtained by the hexamethylenediamine salt reaction with ¹³ C labeled represented by the structural formula B-2 is selected from the polymer .

Structural Formula A-5 Structural Formula B-2

Structure A-6 Structure B-2
Provided that X in the structural formula B-2 is a halogen or a monovalent anion. A composition for an internal standard for analyzing the oligomer content of a guanidine polymer in a household article, comprising the polymer of any one of claims 1 to 7. A method for analyzing the oligomer content of a guanidine polymer in a household article through a mass spectrometry method using the composition for an internal standard material according to claim 8. 10. The method of claim 9,
A method for analyzing the oligomer content of a guanidine polymer in a commodity, wherein the mass spectrometry method uses MAI DI-TOF mass spectrometry. a) extracting a sample solution containing an oligomer of a guanidine based polymer from a household article;
b) preparing a sample by varying the relative concentration of the sample solution and the composition for the internal standard material by mixing the extracted sample solution with the composition for the internal standard material of claim 8, and preparing a sample from each of the MALDI-TOF desorption / ionization time-of-flight mass spectrometry spectrum; And
c) quantitatively analyzing the oligomer content of the guanidine polymer in the sample solution by obtaining a calibration curve using each of the mass spectrometry spectra, and analyzing the oligomer content of the guanidine polymer in the household article . 12. The method of claim 11,
(A-1) between the steps a) and b), the step of purifying the sample using the solid phase extraction cartridge (SPE) A method for analyzing the oligomer content of a guanidine polymer. 13. The method of claim 12,
Wherein the solid phase extractor (SPE) is a Mixed-mode Cation Exchange (MCX) cartridge. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; 12. The method of claim 11,
Wherein the mass spectrometry spectrum in the step b) is obtained by mixing a sample obtained by changing the relative concentration and an ionic liquid to obtain an ionic liquid matrix, and analyzing the oligomer content of the guanidine polymer in the household article Way. 12. The method of claim 11,
The method for analyzing the oligomer content of a guanidine polymer in a household article, wherein the household article is any one selected from the group consisting of a wet tissue, a contact lens cleaning liquid, a make-up remover, a hand rinse liquid, a bactericide, a deodorant and a fragrance. A method for analyzing an oligomer content of a polymer contained in a detection target,
a) extracting a sample solution containing an oligomer of a polymeric polymer from a detection target;
b) preparing a sample by varying the relative concentration of the sample solution and the composition for the internal standard material by mixing the extracted sample solution with a radioisotope labeled composition for an internal standard substance, and preparing a sample from each MALDI-TOF (matrix -assisted laser desorption / ionization time-of-flight mass spectrometry spectra; And
c) quantitatively analyzing the oligomer content of the polymer in the sample solution by obtaining a calibration curve using each of the mass spectrometry spectra, and analyzing the oligomer content of the polymer contained in the detection target . 15. The method of claim 14,
Wherein the composition for the internal standard substance is characterized in that the carbon atom or the nitrogen atom in the molecule of the reference substance is labeled with a radioactive isotope. 17. The method of claim 16,
The mass spectrometry spectrum in step b) is obtained by mixing a sample obtained by changing the relative concentration and an ionic liquid and using the ionic liquid matrix to analyze the oligomer content of the polymer contained in the object to be detected How to.

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